Disclosed herein are devices and methods for measuring reagent volumes and determining the accuracy of liquid dispensers used in biochemical and biomedical assays. More particularly, disclosed herein are devices used with robotic equipment in the processing of microtiter-plate based assays. The devices and methods permit verification of the accuracy of the dispensing portion of diagnostic instruments in the analysis of samples.
The development of microtiter-plate methods allows the processing of a large number of samples simultaneously. Several laboratory and robotic systems have been developed for the purpose of processing microtiter plates. These devices are designed to increase laboratory throughput and are generally preferred over non-automated processes. Automated systems are more efficient and easier to control with less chance for random procedural errors. Automated systems generate homogeneous assays and have helped to eliminate error-prone washing and transferring steps. Automated systems generally provide for the accurate and precise delivery of assay reagents and other necessary fluids to individual reaction vessels
Automation techniques vary from simple semi-automated liquid handling systems to fully integrated automated systems that include multiple robot arms and pipetting stations. Although automated systems have improved quality and reproducibility of fluid transfer, periodic calibration and quality assurance determinations are required. The relatively small size of the wells on the microtiter plate requires the precise delivery of a minute amount of sample. Inaccuracies in the delivery of samples can lead to erroneous results. In order to safeguard the accuracy of the results, the volume delivered by the automated devices must be routinely verified. Assuring the accuracy of biochemical assays is a critical concern for those in the medical diagnostic arts.
Two common techniques for checking the precision and accuracy of fluid dispensers are gravimetric and spectrophotometric techniques. The gravimetric technique is based on the weight of a pure sample of water dispensed by the device. The water is weighed using a balance calibrated with NIST-traceable weights. The actual dispensed volume is calculated from the measured weight and the density, taking into account temperature and evaporation rate. The proper type of equipment and operating environment which are needed to make gravimetric verification of automated pipetting devices are usually not available in a clinical laboratory and requires a visit by a field service technician to perform the diagnostic tests on site. Precision balances are expensive and difficult to use in the field service environment. They are sensitive to various environmental influences such as proximity to air conditioning vents, vibration and leveling. Precision balances also require careful set-up and regular calibration to ensure proper function. Thus, the skill level and time required to conduct these measurements are very high which makes gravimetric verification impractical in a clinical laboratory.
The spectrophotometric technique of volume verification employs a solution containing a known concentration of a high-colored chromagen. Aliquots of the sample solution are dispensed into a known volume of diluent and the absorbance measured. The actual dispensed volume is then calculated from the absorbance, the light path, the extinction coefficient and the diluent volume. In reality the concentration of the colorimetric reagent, cannot be precisely controlled. Therefore, verification techniques that calculate volume directly from the absorbance measured are often inaccurate. Furthermore, this method of volume verification is a manual procedure that is not performed in coordination with an automated workstation. The method requires additional solutions and is time consuming and prone to human error.